A simple and efficient method of generating HCMV pp65-specific T cells using overlapping peptides.

The methods for expansion of human cytomegalovirus (HCMV)-specific T lymphocytes are limited due to the complex culture process, long culture duration, and human leukocyte antigen (HLA) restriction. Here, we report that in vitro stimulation with pp65 kDa phosphoprotein (pp65)-derived overlapping synthetic peptides rapidly generates large numbers of HCMV-specific cytotoxic T lymphocytes from peripheral blood mononuclear cells (PBMCs) regardless of HLA type. Treatment of PBMCs from healthy volunteers expressing HLA-A*02:01 or HLA-A*24:02 with 138 pp65 overlapping peptides (OLP) resulted in an expansion of HCMV pp65 NLVPMVATV (NLV) pentamer-specific CD8+ T lymphocytes that expressed interferon (IFN)-γ, but the pp65 NLV peptide did not generate HCMV-specific CD8+ T lymphocytes in PBMCs obtained from an HLA-A*24:02 donor due to HLA restriction. The OLP-induced T lymphocytes specific for HCMV derived from PBMCs of HLA-A*02:01- and HLA-A*24:02-expressing donors showed effective cytolytic responses against target cells loaded with OLP or the NLV epitope, but pp65 NLV peptide-induced T lymphocytes did not. Phenotypic analyses demonstrated that OLP increased the frequency of CD3+ CD8+ cells, but not CD3+ CD4+, CD14+, or CD56+ cells, in donor PBMCs. Thus, this study provides evidence that in vitro stimulation with OLP efficiently generates sufficient numbers of HCMV pp65-specific cytotoxic T lymphocytes for adoptive cell therapy. Keywords: human cytomegalovirus; cytotoxic T lymphocyte; overlapping peptides; pp65; cytotoxicity.